We present the DeNovoGear software for analyzing de novo mutations from familial and somatic tissue sequencing data. DeNovoGear uses likelihood-based error modeling to reduce the false positive rate of mutation discovery in exome analysis, and fragment information to identify the parental origin of germline mutations. We used our program to create a whole-genome de novo indel callset with a 95% validation rate, producing a direct estimate of the human germline indel mutation rate.

Gonadal failure, along with early pregnancy loss and perinatal death, may be an important filter that limits the propagation of harmful mutations in the human population. We hypothesized that men with spermatogenic impairment, a disease with unknown genetic architecture and a common cause of male infertility, are enriched for rare deleterious mutations compared to men with normal spermatogenesis. After assaying genomewide SNPs and CNVs in 323 Caucasian men with idiopathic spermatogenic impairment and more than 1,100 controls, we estimate that each rare autosomal deletion detected in our study multiplicatively changes a man's risk of disease by 10% (OR 1.10 [1.04–1.16], p<2×10−3), rare X-linked CNVs by 29%, (OR 1.29 [1.11–1.50], p<1×10−3), and rare Y-linked duplications by 88% (OR 1.88 [1.13–3.13], p<0.03). By contrasting the properties of our case-specific CNVs with those of CNV callsets from cases of autism, schizophrenia, bipolar disorder, and intellectual disability, we propose that the CNV burden in spermatogenic impairment is distinct from the burden of large, dominant mutations described for neurodevelopmental disorders. We identified two patients with deletions of DMRT1, a gene on chromosome 9p24.3 orthologous to the putative sex determination locus of the avian ZW chromosome system. In an independent sample of Han Chinese men, we identified 3 more DMRT1 deletions in 979 cases of idiopathic azoospermia and none in 1,734 controls, and found none in an additional 4,519 controls from public databases. The combined results indicate that DMRT1 loss-of-function mutations are a risk factor and potential genetic cause of human spermatogenic failure (frequency of 0.38% in 1306 cases and 0% in 7,754 controls, p = 6.2×10−5). Our study identifies other recurrent CNVs as potential causes of idiopathic azoospermia and generates hypotheses for directing future studies on the genetic basis of male infertility and IVF outcomes.

The azoospermia factor c (AZFc) region harbors multi-copy genes that are expressed in the testis. Deletions of the AZFc region lead to reduced copy numbers of these genes. Four (partial) AZFc deletions have been described of which the b2/b4 and gr/gr deletions affect semen quality. In most studies, (partial) AZFc deletions are identified and characterized using plus/minus sequence site tag (STS) polymerase chain reaction (PCR). However, secondary duplications increase the gene copy number without re-introducing the STS boundary marker. Consequently, the actual copy number of AZFc genes cannot be determined via STS PCR. In the current study, we first set out to determine by quantitative real-time PCR the actual copy number of all AZFc genes in men with (partial) AZFc deletions based on STS PCR. We then analyzed whether reduced gene copy numbers of each AZFc gene family were associated with reduced total motile sperm count (TMC), regardless of the type of deletion. We screened 840 men and identified 31 unrelated men with (partial) deletions of AZFc based on STS PCR. Of these 31 men, 6 men (19%) had one or more secondary duplications. For all AZFc genes, we found an association between a reduction in the copy number of each individual AZFc gene and reduced TMC. In gr/gr-deleted men, restoration of reduced gene copy numbers restored their TMC to normal values. Our findings suggest that the gene content of the AZFc region has been preserved throughout evolution through a dosage effect of the AZFc genes on TMC safeguarding male fertility.

No abstract